Eomesodermin partners with MITF and PU.1 in myeloid precursors to modulate expression of osteoclast lineage determining transcription factors
Immunoprecipitation with FLAG-MITF, HA-PU.1, and V5-EOMES in HEK-293 cells.
Steps to reproduce
For IP experiments, different combinations of FLAG-MITF, HA-PU.1, and V5-EOMES were transfected into HEK-293 cells. Preparation of cell lysates, IP, and Western blot analysis were as previously described (Carey et al., 2016). Briefly, 200 μg of protein was incubated with 1) anti‐FLAG (Sigma-Aldrich Cat #1804); 2) anti-HA (Cell Signaling Cat #3724); or 3) anti-V5 (Sigma-Aldrich Cat #V8012) antibody overnight at 4°C while shaken. Immunoprecipitated proteins were recovered, washed in lysis buffer, and immunocomplexes released with 2X SDS sample buffer.